Study of sulfate-reducing ammonium oxidation process and its microbial community composition

In most other studies of SRAO (Fdz-Polanco et al. 2001c; Liu et al. 2008; Yang et al. 2009; Jing et al. 2010), NO₂⁻ was found in the effluent, and their stoichiometric ratios were in accordance with Equation (1) or only slightly different, with the only notable exception being the experiments reported by Rikmann et al. (2012) and Sabumon (2008). Recent studies showed disproportionally higher removals of ammonium. At the same time, it should be noted that in most analyses the effluent contained more NO₃⁻ than the influent (Mahmood et al. 2007; Sabumon 2008; Rikmann et al. 2012).

In the simultaneous removal of ammonium and sulfate, n(NH₄⁺-N)/n(SO₄²⁻) gave results of between 0.7 and 2 (Sabumon 2007; Liu et al. 2008; Yang et al. 2009; Rikmann et al. 2012), which made the mechanism of SRAO process unclear and complex (Zhang et al. 2013). Many researchers reached different conclusions in studying NO₃⁻ and NO₂⁻, but they were all analysed from the angles of matrix concentrations and product changes. Few studies have focused on microbial community changes. Liu et al. (2008) isolated and described a new autotrophic Planctomycetes bacterium, provisionally named Anammoxoglobus sulfate. SRAO was one of many possible metabolic pathways for this particular bacterium, with other pathways including the utilization of various NO₂⁻ and NO₃⁻ compounds. Hence, the SRAO process might be a more common metabolic pathway in nature and therefore may not be restricted to a few genera of bacteria.

This work adopts a self-designed anaerobic biological reactor, using ammonium and sulfate as the substrate to start SRAO, to explore wastewater treatment performance and the effect of various environmental factors. The end products of the SRAO reaction were tested, and the microbial community was analysed to explain the mechanism. Moreover, the N/S ratio was explored to describe the structure of the products. A determination and observation of the SRAO process was carried out for the comprehensive treatment of wastewaters containing high levels of ammonium and sulfate.

As illustrated in Figure 1, a self-designed circulating flow reactor was used in this study. The reactor contained a round column made of plexiglas, which were 80 cm in height and 3 L in volume. Non-woven fabrics (100 cm × 12 cm) were fixed on the right side of the reactor to enhance microbial growth. On the left side, there was an axial agitator rotating at 120 rpm, installed to mix the substrates evenly. To maintain anaerobic conditions, nitrogen was flushed into the reactor for 10 min before every test. The reactor was kept at 30 ± 1 °C using a water bath and was covered by a black cloth to protect the bacteria from light and algal growth (Liu et al. 2012). The inlet was at the top of the reactor and was used to introduce influent into the tank. The outlet of the silicone tube was located at the bottom of the reactor, and discharge was released from the outlet.

The reactor was seeded with 0.8 L mixed sludge, which consisted of 0.2 L anaerobic granular sludge that was obtained from a municipal wastewater plant and 0.6 L denitrification sludge from a continuous stirred-tank reactor (CSTR). The volatile suspended solids (VSS) of the seed sludge was 3.0 g·L⁻¹.

Inorganic synthetic wastewater including ammonium and sulfate was the main source for the microorganisms as well as trace elements that were introduced as the influent to the reactor. Ammonium and sulfate were added at a molar ratio of 2:1 and 4:1, respectively, in the form of (NH₄)₂SO₄ and NH₄Cl, respectively. The pH of the reactor was maintained at 8.1–8.6 by adding Na₂CO₃ and KHCO₃, which also served as the bicarbonate source. The composition of the mineral medium was based on that of Yuan et al. (2013).

NH₄⁺-N, SO₄²⁻, NO₂^–N and NO₃^–N were measured using an ion chromatograph (ICS-2000, DIONEX). The pH was measured with a pH meter (FE28, METTLER). The concentration of sulfide, including HS⁻, S²⁻ and H₂S_(aq), was measured by the methylene blue method. All liquid samples were filtered through a 0.45 μm membrane before analysis.

The activated sludge samples were taken from the reactor by using centrifuge tubes (10 mL) on days 62 and 187 and were stored at −20 °C. A total DNA extraction (2 μL) from the samples was performed using a PowerSoil DNA Isolation Kit (MoBio, USA) according to the instructions (Zhang et al. 2009). The quality of the extract DNA was examined by electrophoresis using a 1% agarose gel, and the concentration was tested with a UV-V is spectrophotometer (NanoDrop 2000, USA). The V3-V5 region of the 16S rRNA gene was amplified using the bacterial primers 515F (GTGCCAGCMGCCGCGG) and 907R (CCGTCAATTCMTTTRAGTTT), and polymerase chain reaction (PCR) amplification was performed according to Chen et al. (2010). The purified amplicon was quantified by a QuantiFluor-ST Fluorometer (Promega, USA) and the PCR results were analysed on an Illumina MiSeq platform at Major Bio-Pharm Technology Co, Ltd (Shanghai, China). Trimmomatic and FLASH methods were used for the sequencing data analysis.

The concentration change of each matrix during the operation of the reactor is shown in Figure 2. The seed sludge was fed with synthetic wastewater at an NH₄⁺-N concentration of 50.0 mg·L⁻¹, an NH₄⁺-N to SO₄^2–S molar ratio (N/S) of 2, a hydraulic retention time (HRT) of 48 hours, a pH of 8.1–8.6, and a temperature of 30 ± 1 °C for the first 26 days, when some NH₄⁺ and SO₄²⁻ were converted and some heterotrophic microorganisms began to die due to the difference in environmental conditions. The removal of NH₄⁺ and SO₄²⁻ was clearly observed as the experiment continued. After 62 days, the HRT was decreased to 24 hours and the concentration of NH₄⁺-N was gradually increased to 120.0 mg·L⁻¹ to maintain an N/S of 2. The removal efficiencies of NH₄⁺-N and SO₄^2–S were up to 92.0% and 30.5%, respectively, and the minimum concentrations achieved in the effluent were 8.4 mg·L⁻¹ and 67.3 mg·L⁻¹, respectively. NO₂⁻ and NO₃⁻ could be observed in the effluent at the average concentrations of 4.5 mg·L⁻¹ and 9.6 mg·L⁻¹, respectively. In this reactor, after being inoculated with inorganic synthetic wastewater and flushed with nitrogen before testing, the NO₂⁻ and NO₃⁻ were excluded during the nitrification process; thus, only SO₄²⁻ acted as an electron donor to NH₄⁺. From days 188 to 213, the influent NH₄⁺-N concentration was maintained at 180.0 mg·L⁻¹ and the SO₄^2–S concentration was 360 mg·L⁻¹. A clear decline in the NH₄⁺ and SO₄²⁻ removal efficiencies appeared. The NH₄⁺-N removal efficiency in this period decreased from 59.2% to 23.4% and was stable at approximately 20% during the rest of the experiment. The SO₄^2–S in the effluent increased from approximately 66.7 mg·L⁻¹ to 111.5 mg·L⁻¹, and then to 269.8 mg·L⁻¹ after increasing the influent SO₄²⁻ concentration. At the end of the experiment, SO₄²⁻ was hardly converted. During the whole operation, sulfide from the effluent was detected only at the early stage. As a result, sulfate-reducing bacteria used the remaining organics to reduce SO₄²⁻ in the reactor.

The sludge was observed to change from black to yellow in the reactor, as compared to the small amount of yellow granular effluent (Figure 3). We determined whether the yellow matter was S⁰ by using organic solvents as referenced in Liu et al. (2015b). The result showed that S⁰ was produced during the process.

At an HRT of 24 hours and with N/S ratios of 2:1 and 4:1, three levels of NH₄⁺-N concentrations were investigated in the reactor (Figure 4(a) and 4(b)). At influent concentration of approximately 118 mg·L⁻¹, the NH₄⁺-N removal efficiency at an N/S of 2:1 was 56.4%, which was lower than that at an N/S of 4:1. The nitrogen conversion was 44.8% when the N/S ratio was 4:1, which was also higher than at the N/S ratio of 2:1. However, approximately 16.7% of NO₂⁻ was produced when the N/S ratio was 2:1. When the N/S ratio was 4:1, the NO₂⁻ yield was 22.6% and the effluent concentration was 10.46 mg/L, which might be toxic to microorganisms in the reactor. At an influent NH₄⁺-N concentration of approximately 178 mg·L⁻¹, a regular pattern that improved the influent N/S ratio enhanced the NH₄⁺-N removal efficiency and nitrogen conversion but accumulated more NO₂⁻. When the N/S ratio was 2:1, the NO₃⁻ product was approximately 7.2%, although the product was far more than when the N/S was 4.

In contrast with these two situations, when the N/S of the influent was improved, the NH₄⁺-N in the conversion ratio was higher and the NO₂⁻ formation was higher. However, the NO₃⁻ formation was lower. The scenario described above also applied to high levels of substrates.

According to Figure 4(c), there was no apparent N/S permanent conversion ratio, which was between 2.1 and 12.9, meaning that the sulfate-reducing anammox process was a multi-step reaction.

High-throughput 16S rRNA gene sequencing technology was used to identify the microbial communities at influent NH₄⁺-N concentrations of 50 mg·L⁻¹ and 120 mg·L⁻¹. The phylogenetic relationship and classification of the bacterial 16S rRNA sequence from the sludge samples extracted on days 62 and 187 with relative abundances above 1% are shown by phylum in Figure 5. The results showed that Proteobacteria (39.9%, 30.9%) were the most abundant, followed by Chloroflexi (11.1%, 16.1%), Bacteroidetes (10.4%, 13.9%), Chlorobi (7.8%,14.5), Acidobacteria (5.3%, 5.2%) and Planctomycetes (3.2%, 4.4%) in the two samples. The conclusion was that the dominant bacterial communities were comparatively stable, but their relative abundances changed over the operation of the reactor. Relative abundance increases in Acidobacteria, Nitrospirae, Armatimonadetes and Proteobacteria observed in the samples could indicate that bacteria played a more important role in high ammonium and sulfate environments than in low ammonium and sulfate environments. Acidobacteria could use multiple electron acceptors to reduce iron or single carbon compounds. Nitrospirae was previously identified as the dominant functional bacteria in several anammox reactors used for nitrogen removal (Zhang et al. 2013). Proteobacteria had a more versatile metabolism. The large proportion of Proteobacteria and Armatimonadetes present in this reactor contributed to the partial nitrification process and NO₂⁻ accumulation (Liu et al. 2012; Mi et al. 2017). Thus, the SRAO process could be a more common metabolic pathway in nature rather than just being restricted to a few genera of bacteria.

Moreover, no traditional sulfate-reducing bacteria, such as Sulfurimonas (Takai 2004) and Desulfobacter, were found in this reactor, thus indicating that sulphur removal was carried out by other bacteria.

Sludge samples extracted from the reactor were used to identify the microbial community structures when the 62 and 187 day samples contained ammonium oxidation bacteria, denitrifying bacteria and anaerobic ammonium oxidation bacteria (Table 1). The results provided a comprehensive and deeper insight into the microbial community compositions in the different influent concentrations, which could be valuable for studying NO₃⁻ and NO₂⁻ sources. The increase in NH₄⁺-N oxidizing bacteria (AOB), known as a type of chemolithotroph that could convert NH₄⁺-N to NO₂⁻, included Nitrosomonas and Nitrosomonadaceae, which were also responsible for part of the NO₂⁻ produced by the ammonium-oxidizing process. Representing a miniscule portion of the NO₂⁻ oxidizing bacteria (NOB), only Nitrospira was detected in the sludge samples. NO₂⁻ can be oxidized to NO₃⁻ to prove that there is a certain amount of NO₃⁻ in the reactor. Since NH₄⁺-N had a relative high removal efficiency and there was a small amount of AOB, there must have been a novel way to consume ammonium during the process.

The potential functional bacteria that oxidized ammonium belong to Planctomycetes including Pla4_lineage, SM1A02, and Planctomycetes, whose proportion increased from 1.5% to 3.0%. The bacteria of Planctomycetes certainly constituted a new genus in the anammox line, but there is no related research that analyses their biochemical performance.

Approximately 12.4% of the denitrifying bacteria were found in sludge sample 2, which showed a 1.2% increase compared to sample 1. The results showed that part of nitrogen was converted by the nitrification-denitrification process. Traditional denitrification occurs simultaneously with SRAO, which leads to an immense boost in SRAO functional bacteria and thus improves the removal efficiency of ammonium and sulfate.

Simultaneous reduction of ammonium and sulfate was achieved in a self-designed reactor; the removal efficiency of NH₄⁺-N was 92.0% and that of SO₄^2–S was 59.2%. NO₂⁻ and NO₃⁻ were found in substantial amounts in the effluent of the reactor. A higher influent N/S ratio could increase the N/S conversion ratio and accelerate NO₂⁻ and nitrogen production. The sludge acclimated in the reactor had Proteobacteria, Chloroflexi, Bacteroidetes, Chlorobi, Acidobacteria and Planctomycetes after the reactor had run for 187 days. Three kinds of Planctomycetes were found: Pla4_lineage, SM1A02, Planctomycetaceae; all were found to have increased their percentages compared to the initial amount. Since denitrifying bacteria and AOB had also acclimated after the operation of the reactor, traditional denitrification was observed to be occurring simultaneously with SRAO, which offers a substantial biotechnological potential for the complete removal of ammonium and sulfate, and should therefore be used more widely. Future work will focus on isolating the functional bacteria and avoiding the production of excess nitrate and nitrite.

This study was supported by the China Environmental Protection Foundation, GePing Green Action, Liaoning Environmental Research and the Education Fund ‘123 Project’ (Grant No. CEPF2014-123-2-6).